The present invention relates to a material for activating adhesion, multiplication and cell-layer formation of epidermal cells of human and mammalian types.
The present active material is useful as an epidermal cell-culture bed and a defect skin repairing material or wound coating material for vital tissue.
Cells are capable of obviously or latently self-multiplying as an essential unit of life, while each of organogenetic cells in multicellular organisms differentiates into various functional cells in a predetermined manner of multiplication.
In the case of the epidermis which protects an organism from the outside environment, cells are formed as a laminar layer to cover dermis situating under the epidermis and multiply only by adhering to other vital tissues or artificial materials to form a planar layer.
More specifically, the human epidermis comprises several layers and unevenly comes in contact with the dermis under the cellular layers through a basal layer so that the epidermis has a larger contact surface to maintain a close relationship with the dermis.
The epidermis has a so-called sebaceous membrane as a thin outermost lipid layer which functions to prevent water from evaporating, and under the membrane there are water-containing keratinous layers mainly consisting of keratin.
Each cell is arranged as a layer under the keratinous layer to form a cellular layer which comes in contact with a basal layer.
These cells repeatedly divide on the basal layer to develop new cells. That is, the basal layer is a base where new cells are formed and developed.
Epidermal cells thus formed in the basal layer gradually rise to the surface and then die when they arrive at the skin surface, thereby cell nuclei disappearing to form a keratinous layer.
As described above, the basal layer is located in the innermost part of the epidermis as a complicatedly uneven layer to form the contact surface so as to keep a close relationship with the epidermal cellular layer.
Although the formation and multiplication of epidermal cells on the basal layer was unsuccessful for reproduction in vitro for a long time, it has recently been possible to conduct the in vitro multiplication thereof because various kinds of solid surface base-beds for adhering epidermal cells were developed.
Even in such an in vitro multiplication, however, it has been quite difficult to form a plain cellular layer similar to that of in vivo multiplication for the lack of mutual adhesion of the cells.
Synthetic high polymer materials, such as polystyrene, natural high polymer materials such as collagen, and synthetic high polymer materials coated with natural materials have been conventionally developed as a cell culture bed. However, as these materials are not always the multiplication of epidermal cells, development of a novel cell culture bed has been desired.
Recently, there was proposed a cell culture bed comprising a water-insoluble silk membrane as a natural high polymer (see, JP-A No. 4-41,595).
The above mentioned cell culture bed is a xcex2-type silk membrane prepared by crystallizing an amorphous silk membrane with an alcohol to water-insolubilize it, and which the animal cellular adhesion rate is improved compared with a conventional polystyrene culture bed.
As a matter of course, improved adherence to the bed is essentially necessary to epidermal cell cultures, while it is also required to form a plain cellular layer in which adjacent cells strongly adhere with each other.
Particularly, to apply such an epidermal cell culture technique to a wound healing method, it is still expected to develop a culture bed which promotes formation of a plain cellular layer.
Accordingly, it is an object of the present invention to provide a novel material for activating epidermal cell multiplication in which are effective adhesion and multiplication of human and mammalian epidermal cells to a base-bed surface obtained but also intercellular adhesion is promoted to form a plain cellular layer.
A further object of the present invention is to provide a novel culture bed in which in vitro formation of human and mammalian epidermal cellular layers can be conducted.
A still further object of the present invention is to provide a wound coating material for healing a skin defect region or wound of humans and mammals.
Another object of the present invention is to contribute to the basic study of epidermal functions by conducting the in vitro development of epidermal cells and also to the investigation of the action mechanism of skin defect healing by means of a novel material for activating epidermal cell multiplication.
The inventors have investigated in detail the interaction between crystal form of silk protein and epidermal cells and finally found that silk protein of a crystal form having a backbone helix structure, and especially xcex1-type or xcex1-helix silk fibroin is an outstanding material for achieving the above mentioned objects.
The present invention is based on this information and provides a material for activating epidermal cell multiplication, an epidermal cell culture bed in which the present material is used as a culture bed for cell multiplication, and a wound coating material in which the present material is used on the wound.
According to the present invention, there is provided (1) a material for activating epidermal cell multiplication which comprises silk fibroin of the xcex1-type or having an xcex1-helix X-ray diffraction pattern as a principal constituent.
There is also provided (2) a material for activating epidermal cell multiplication which comprises silk fibroin of xcex1-type or having an xcex1-helix X-ray diffraction pattern as a principal constituent and 50% by weight or less of a synthetic polyamino acid of an xcex1-helix crystalline form.
There is also provided (3) a material for activating epidermal cell multiplication described in item (1) or (2) in which said material is in the form of a fiber, membrane or powder.
There is also provided (4) a material for activating epidermal cell multiplication in which a solid surface of organic or inorganic substances is coated with a coating containing silk fibroin of xcex1-type or having an xcex1-helix X-ray diffraction pattern as a principal constituent.
There is also provided (5) an epidermal cell culture bed which comprises a coating containing silk fibroin of xcex1-type or having an xcex1-helix X-ray diffraction pattern as a principal constituent.
There is also provided (6) an epidermal cell culture bed in which a solid surface of organic or inorganic substances is coated with a stuff containing silk fibroin of xcex1-type or xcex1-helix X-ray diffraction pattern as a principal constituent.
There is also provided (7) an epidermal cell culture bed described in item (5) or (6) in which said bed is in the form of a membrane.
There is provided (8) a wound coating material which comprises a silk fibroin of xcex1-type or having xcex1-helix X-ray diffraction pattern as a principal constituent.
There is also provided (9) a wound coating material which comprises silk fibroin of xcex1-type or having an xcex1-helix X-ray diffraction pattern as a principal constituent and 50% by weight or less of a synthetic polyamino acid of xcex1-helix crystalline form.
There is also provided (10) a wound coating material described in item (8) or (9) in which said material is in the form of a fiber, membrane or powder.
There is also provided (11) a wound coating material in which a solid surface of organic or inorganic substances is coated with a coating containing silk fibroin of xcex1-type or having an xcex1-helix X-ray diffraction pattern as a principal constituent.
A material for activating epidermal cell multiplication of the present invention is a material in which human and mammalian epidermal cells effectively adhere to a basal surface and multiply while forming a plain layer as a result of intercellular adhesion, i.e., a material in which adhesion and multiplication of epidermal cells as well as formation of a multicellular layer can be promoted.
A concept of the present invention also includes an epidermal cell culturing bed and a wound coating material.
Further, the present material is not limited to a specific shape or structure and may be in the form of a membrane such as sheet or film, powder, bead, fiber, etc.
All of the following may be used as raw materials for preparing a material for activating epidermal cell multiplication of the present invention:
a) Raw materials selected from cocoon yarn, raw silk yarn, silk fabric, silk yarn (fibroin fiber) or untreated products thereof;
b) Fibroin or sericin independently separated and prepared from cocoon yarn, raw silk yarn, silk fabric, silk yarn or untreated products thereof;
c) Raw materials prepared by mixing a material selected from cocoon yarn, raw silk yarn, silk fabric, silk yarn or untreated products thereof and independently separated and prepared fibroin or serine; and
d) Protein fibrous materials produced by silkworms including domestic and wild-silkworms, such as, for example, used products of sericin- or fibroin-containing fiber, powder, film, etc., and liquid silk contained in a silk worm.
The amount of sericin contained in these raw materials is preferably 40% or less, and more preferably 20% or less from the standpoint of phase separation.
Further, not more than 50% by weight of a synthetic polyamino acid having a helical structure may be mixed with the above mentioned raw materials from the standpoint of formability. Such a polyamino acid includes those materials which have a backbone repeating unit of xe2x80x94Axe2x80x94, xe2x80x94Axe2x80x94Gxe2x80x94, xe2x80x94Axe2x80x94Gxe2x80x94Sxe2x80x94, xe2x80x94Axe2x80x94Gxe2x80x94Axe2x80x94Gxe2x80x94Sxe2x80x94, etc., wherein A, G and S represent alanine, glycine and serine, respectively. When independently prepared fibroin is used as a raw material, for example, the above mentioned synthetic polyamino acid having a helical structure may be mixed thereto in an amount of not more than 50% by weight.
A raw material used in the present invention, containing silk fibroin as a principal constituent, is a material in which silk fibroin is converted to xcex1-type or has an xcex1-helix structure by an xcex1-crystallizing process as will be described in the following.
xcex1-crystallization
Fibroin- and sericin-containing raw materials are degummed, dissolved in an aqueous solvent in the presence of a neutral salt and desalted to yield an aqueous fibroin solution, which is then cast on a smooth solid surface and dried to form a water soluble dry silk membrane.
Then, in order to convert the thus formed membrane to be water insoluble and in the form of a sheet or film having a crystal form of xcex1-type or an xcex1-helix structure, environmental moisture (for example, relative humidity of about 80 to 90%) is controlled to maintain a water content within 40xc2x115% at room temperature and left to stand for such a long period of time as 12 to 24 hours. More specifically, the above mentioned process is conducted as in the following.
1) Crystallization in the course of drying
The aqueous fibroin solution is cast on a smooth solid surface and then dried to a water content of 40xc2x115% in a drying step, which is kept for a long period of time under this condition.
2) Crystallization after drying
The aqueous fibroin solution is dried and kept as it is at room temperature and a water content of 40xc2x115% for a long period of time.
The thus prepared water insoluble silk membrane of xcex1-type or having an xcex1-helix structure is dried and powdered to form a powdery material.
The thickness of the silk membrane is an important factor. If the silk membrane is comparatively thin, the molecular structure thereof is affected by a substrate material and does not always results in satisfied crystalline form. If the membrane is rather thick, on the other hand, it is apt to release from the substrate material.
From the above mentioned viewpoint, the silk membrane is preferably 0.05 to 20 xcexcm, and more preferably 0.5 to 10 xcexcm in thickness.
According to the present invention, it is possible to provide a greatly improved material which has greatly improved properties as essential requirements for activating epidermal cells, such as cell adhesion, multiplicity, intercellular adhesion, stretchability of cellular layer, etc.
It is therefore expected that the present invention can considerably contribute to the solution of the main problems in epidermal studies, for example, a biochemical reaction occurring when an epidermal cellular layer is formed, or to application of epidermal cell culture technology for care of wound skin.